Journal: Acta biochimica et biophysica Sinica

Article Title: Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

doi: 10.3724/abbs.2024141

Figure Lengend Snippet: Figure 3. VAC alleviated the renal inflammatory response and oxidative stress in T2DM mice (A) MDA contents in diabetic kidney tissues. n=6. (B) GSH-Px activity in diabetic kidney tissues. n=6. (C) Averaged fluorescence intensity of DHE fluorescence in diabetic kidney tissues. n=6. (D) Averaged fluorescence intensity of DCFH-DA fluorescence staining of diabetic kidney tissues. n=6. (E) DHE fluorescence staining or DCFH-DA fluorescence staining of diabetic kidney tissues. Scale bar: 100 μm. (F) F4/80 staining of diabetic kidney tissues. Scale bar: 50 μm, and the average fluorescence intensity of F4/80-expressing diabetic kidney tissues is shown. (G‒M) Representative blot images and quantitative analysis of phosphorylated NFκB P65, Nrf2, catalase, SOD3, SOD2 and SOD1. n=4. *P<0.05, **P<0.01, ***P<0.001 vs Ctrl. #P<0.05, ##P<0.01, ###P<0.001 vs DN.

Article Snippet: Primary antibodies against catalase, SOD1, SOD2 and SOD3 were procured from Boster Biological Technology (Wuhan, China).

Techniques: Activity Assay, Fluorescence, Staining, Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

doi: 10.3724/abbs.2024141

Figure Lengend Snippet: Figure 5. VAC alleviated the inflammatory response and oxidative stress in diabetic kidneys HK-2 cells were preincubated with 5 μM VAC for 12 h, followed by exposure to 35 mM HG for 48 h. (A–C) Relative mRNA levels of IL-1β, VCAM-1 and COX2. (D-F) DHE fluorescence staining or DCFH-DA fluorescence staining of HK-2 cells. Scale bar: 100 μm. (G–K) Relative mRNA levels of Nrf2, catalase, SOD3, SOD2 and SOD1. (L–R) Representative blot images and quantitative analysis of phosphorylated NFκB P65, Nrf2, catalase, SOD3, SOD2 and SOD1. *P<0.05, **P<0.01, ***P<0.001 vs NG. #P<0.05, ##P<0.01, ###P<0.001 vs HG. n=4.

Article Snippet: Primary antibodies against catalase, SOD1, SOD2 and SOD3 were procured from Boster Biological Technology (Wuhan, China).

Techniques: Fluorescence, Staining

Journal: Acta biochimica et biophysica Sinica

Article Title: Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

doi: 10.3724/abbs.2024141

Figure Lengend Snippet: Figure 8. VAC ameliorated fibrosis, the inflammatory response and oxidative stress in HG-induced HK-2 cells exposed to the EGFR inhibitor AG1478 or the ERK inhibitor U0126 (A–D) Relative mRNA levels of collagen-1, TGF-β1, α-SMA and E-cadherin in HK-2 cells. (E–G) Relative mRNA levels of IL-1β, VCAM-1 and COX-2 in HK-2 cells. (H) Averaged fluorescence intensity of DHE fluorescence in HK-2 cells. (I) DHE staining was performed on HG-induced HK-2 cells. Scale bar: 100 μm. (J–P) Representative blot images and quantitative analysis of phosphorylated and total NFκB P65, Nrf2, catalase, SOD3, SOD2 and SOD1. *P<0.05, **P<0.01, ***P<0.001 vs NG. #P<0.05, ##P<0.01, ###P<0.001 vs HG. n=4.

Article Snippet: Primary antibodies against catalase, SOD1, SOD2 and SOD3 were procured from Boster Biological Technology (Wuhan, China).

Techniques: Fluorescence, Staining

Journal: Veterinary Medicine and Science

Article Title: Expression of xenobiotic metabolising enzymes in lungs of horses with or without histological evidence of lower airway inflammation

doi: 10.1002/vms3.331

Figure Lengend Snippet: Accession numbers and primer sequences for CYP1A1, CYP2A13, GSTM1, GSTP1, SOD3 and GAPDH respectively

Article Snippet: Primary SOD3 rabbit anti‐human/mouse/rat , Biosite, USA , 1:3,000.

Techniques:

Journal: Veterinary Medicine and Science

Article Title: Expression of xenobiotic metabolising enzymes in lungs of horses with or without histological evidence of lower airway inflammation

doi: 10.1002/vms3.331

Figure Lengend Snippet: Polyclonal antibodies used in the immunohistochemistry

Article Snippet: Primary SOD3 rabbit anti‐human/mouse/rat , Biosite, USA , 1:3,000.

Techniques: Plasmid Preparation

Journal: Veterinary Medicine and Science

Article Title: Expression of xenobiotic metabolising enzymes in lungs of horses with or without histological evidence of lower airway inflammation

doi: 10.1002/vms3.331

Figure Lengend Snippet: Box plots showing the level of gene expression of cytochrome P450 (CYP) enzymes (a) CYP1A1 and (b) CYP2A13, and enzymes involved in defence against toxic metabolites glutathione‐S‐transferases, (c) GSTM1, (d) GSTP1 and superoxide dismutase, (e) SOD3 in the lungs of horses without (Histo−) or with (Histo+) histological evidence of lower airway inflammation respectively. Gene expression is related to expression of the reference gene GAPDH (according to Schmittgen & Livak, ). The central horizontal line in each box represents the median, whereas the top and bottom lines represent the 25th and 75th percentiles. Whiskers indicate the min and max. p ‐values are shown above each enzyme, a p < .05 indicate a significant difference between horses without or with histological evidence of lower airway inflammation

Article Snippet: Primary SOD3 rabbit anti‐human/mouse/rat , Biosite, USA , 1:3,000.

Techniques: Gene Expression, Expressing

Journal: Veterinary Medicine and Science

Article Title: Expression of xenobiotic metabolising enzymes in lungs of horses with or without histological evidence of lower airway inflammation

doi: 10.1002/vms3.331

Figure Lengend Snippet: Immunohistochemical staining (brown) of (a) CYP1A, (b) SOD3, (c) GSTM1 and (d) GSTP1 in paraffin sections of horse lung tissue. ec, epithelial cells; lbs, lumen of bronchus; lb, lumen of bronchioles; lp, lamina propria; lrb, lumen of respiratory bronchioles; ltb, lumen of terminal bronchioles. Bars = 50 µm

Article Snippet: Primary SOD3 rabbit anti‐human/mouse/rat , Biosite, USA , 1:3,000.

Techniques: Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: SOD3 suppresses early cellular immune responses to parasite infection

doi: 10.1038/s41467-024-49348-0

Figure Lengend Snippet: a Progressively increased expression of SOD3 was observed during P. y. yoelii YM infection. The mouse illustration was created with http://BioRender.com (publishing license: CZ26UIGFUP). b Serum SOD3 expression was elevated in mice after infection with P. berghei ANKA, with peaks observed after 5 days and 9 days. Five biological replicates were included in the analysis; healthy controls were three biological replicates. Statistical significance tested by one-way ANOVA, and multiple comparisons to each day were corrected using Dunnet’s method. WT mice of average survival time was 9 days from the time of challenge. c SOD3 in the sera of P. falciparum –infected patients from the China-Myanmar border (endemic areas, n = 20), other endemic areas (cases were travel-related, n = 24) and healthy donors (endemic and nonendemic areas, n = 41) as measured by enzyme-linked immunosorbent assay (ELISA). SOD3 levels were significantly elevated in malaria patients from all areas compared with corresponding healthy donors. Two-tailed student’s t test was used to test for significant differences between two groups. d SOD3 −/− mice (n = 20) showed significant extended survival time than the WT littermates ( n = 20) after P. y. yoelii YM infection. Survived mice in SOD3 −/− group were humanely euthanized accordingly to the Institutional Animal Care and Use Committee-approved criteria. e Knockout of SOD3 extended the survival of P. y. yoelii 17XL-infected mice, n = 10. The survived mice in SOD3 −/− group were humanely euthanized accordingly to approved criteria. f Knockout of SOD3 decreased parasitemia in both seven and eight days after T. b. brucei infection. Two-tailed student’s t test was used to test for significant differences between two groups. g Knockout of SOD3 extended the survival of T. gondii -infected mice. The survived mice in SOD3 −/− group were humanely euthanized accordingly to approved criteria. Kaplan‒Meier survival curves were calculated using the survival time for each mouse in all groups, and significance was determined by the log-rank test. For d-f, n indicates mouse numbers in graphs. Histograms present the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Brain sections were blocked with 10% FBS in PBS, incubated with anti-SOD3 antibodies (Affinity Biosciences, Cat# DF7753, RRID: AB2841219), incubated with a corresponding secondary antibody, and then stained with 3,3-diaminobenzidine.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Knock-Out

Journal: Nature Communications

Article Title: SOD3 suppresses early cellular immune responses to parasite infection

doi: 10.1038/s41467-024-49348-0

Figure Lengend Snippet: a , b SOD3 is associated with cerebral malaria in WT mice. The accumulation of P. berghei iRBCs was not obvious in the mouse brains of SOD3 −/− mice. However, this phenomenon was reversed in both littermate (WT) and SOD3 o/e mice after infection, which showed severe sequestration of iRBCs in the brains. Overexpression of SOD3 also resulted in increased parasitemia, as reflected by increased luminescence signals in the body. The luminescence images of the mice were recorded with an AniView600 multimode in vivo animal imaging system, n = 3. Statistical tests were two-sided, and Tukey corrected for multiple comparisons. c SOD3 −/− mice displayed extended survival after P. berghei infection. The survived mice in SOD3 −/− group were humanely euthanized accordingly to approved criteria. d SOD3 o/e mice and control mice showed similar vulnerability to the parasite infection. Five biological replicates were used for analysis. Kaplan‒Meier survival curves were calculated using the survival time for each mouse in all groups, and significance was determined by the log-rank test. e Administration of recombinant SOD3 increased the sensitivity of the SOD3 −/− mice to P. berghei infection. For ( c – e ), n indicates mouse numbers in graphs. For c and e, Kaplan-Meier survival curve p -values were performed using Log rank Mantel-COX test. Histograms present the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Brain sections were blocked with 10% FBS in PBS, incubated with anti-SOD3 antibodies (Affinity Biosciences, Cat# DF7753, RRID: AB2841219), incubated with a corresponding secondary antibody, and then stained with 3,3-diaminobenzidine.

Techniques: Infection, Over Expression, In Vivo, Imaging, Control, Recombinant

Journal: Nature Communications

Article Title: SOD3 suppresses early cellular immune responses to parasite infection

doi: 10.1038/s41467-024-49348-0

Figure Lengend Snippet: a SOD3 can bind both T cells and NK cells. Black line indicates the blank control, and the gray line represents omission of the primary antibody as the negative control. The Alexa Fluor™ 488-conjugated goat anti-Rabbit IgG (H + L) secondary antibody was used to recognize the primary antibody. The control sample was stained with a non-specific rabbit IgG as the primary antibody and AF488-goat anti-rabbit IgG secondary antibody. b Depletion of SOD3 in mice rescued the IFN-γ production in the sera ( n = 4). c The expression of IFN-γ protein in the splenic cells of SOD3 −/− mice was much more than that of WT mice after infection illustrated by immunohistochemistry. Scale bar =20 µm. d , e The proportion of IFN-γ-expressing CD8 + T cells was significantly enhanced in SOD3 −/− mice infected with P. y. yoelii (yellow bar). n = 6 ( d ); n = 3 ( e ). f Depletion of IFN-γ in mice resulted in early death in SOD3 −/− mice after P. berghei ANKA infection. Five biological replicates were included in the analysis. Kaplan‒Meier survival curves were calculated using the survival time for each mouse in all groups, and significance was determined by the log-rank test. g SOD3 inhibited T cell activation ( n = 3). h , i SOD3 inhibited the differentiation of Th0 cells (naïve T cells) into effector cells in in vitro experiments ( n = 3). j The proportion of IFN-γ-expressing CD8 + T cells upon stimulation with SOD3 was significantly reduced in in vitro experiments ( n = 3). k, l Western blot and quantitative for JNK and β-actin in cell lysates obtained from T cells sorted from SOD3 −/− and WT mice after infection. The expression of JNK in T cells was significantly lower in WT mice than that in the SOD3 −/− mice. Representative immunoblot using antibodies against β-actin and JNK1/2/3 ( n = 3). m Treatment with SP600125, a selective JNK inhibitor, led to a decrease in the proportion of IFN-γ-expressing CD8 + T cells ( n = 3). t -test two-sided was used to compare change in ( b , d , e , g – j , l , m ). Histograms present the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Brain sections were blocked with 10% FBS in PBS, incubated with anti-SOD3 antibodies (Affinity Biosciences, Cat# DF7753, RRID: AB2841219), incubated with a corresponding secondary antibody, and then stained with 3,3-diaminobenzidine.

Techniques: Control, Negative Control, Staining, Expressing, Infection, Immunohistochemistry, Activation Assay, In Vitro, Western Blot

Journal: Nature Communications

Article Title: SOD3 suppresses early cellular immune responses to parasite infection

doi: 10.1038/s41467-024-49348-0

Figure Lengend Snippet: a IL-2 levels in mouse sera were gradually elevated in SOD3 −/− compared with WT mice after infection by P. berghei ANKA ( n = 5). b – e Statistical analysis of IL-2 production in splenic and circulating CD4 + and CD8 + cells from both WT and SOD3 −/− mice ( n = 6–8) at different time points after P. berghei infection. f Representative flow cytometry plots of splenic IL-2 + CD4 + T cells in both SOD3 −/− mice and WT mice. g , h Statistical analysis of IL-2 production in CD4 + and CD8 + T cells from both WT ( n = 5) and SOD3 −/− mice ( n = 4) after P. y. yoelii infection. i , j Neutralization of IL-2 in SOD3 −/− mice resulted in early death and higher parasitemia after P. berghei infection compared to control mice. Five biological replicates were included in the analysis. Parasitemia was depicted with the area under curve (AUC). The survived mice in SOD3 −/− +saline group and SOD3 −/− +IgG were humanely euthanized accordingly to approved criteria. k Infusion of recombinant IL-2 in WT mice inhibited P. berghei proliferation (n = 5). Parasitemia was depicted with the AUC. Histograms present the mean ± SD. For ( a , g , h ), Kaplan-Meier survival curve p-values were performed using Log rank Mantel-COX test. Source data are provided as a Source Data file.

Article Snippet: Brain sections were blocked with 10% FBS in PBS, incubated with anti-SOD3 antibodies (Affinity Biosciences, Cat# DF7753, RRID: AB2841219), incubated with a corresponding secondary antibody, and then stained with 3,3-diaminobenzidine.

Techniques: Infection, Flow Cytometry, Neutralization, Control, Saline, Recombinant

Journal: Nature Communications

Article Title: SOD3 suppresses early cellular immune responses to parasite infection

doi: 10.1038/s41467-024-49348-0

Figure Lengend Snippet: a , b The proportion of CD8 + or CD4 + T cells producing IL-2 upon treatment with SOD3 was significantly reduced in in vitro experiments, n = 3. c , d Treatment with SP600125, a selective JNK inhibitor, led to a decrease in the proportion of IL-2-producing CD8 + or CD4 + T cells, n = 3. e and f The proportion of IFN-γ + CD122 + T cells but not that of CD122 + T cells expanded in the SOD3 −/− mice, n = 4 ( e , f ). a – f Two-tailed student’s t test was used to test for significant differences between two groups. g Serum levels of IFN-γ was analysis by neutralizing Abs for IL-2, n = 3–4. Histograms present the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Brain sections were blocked with 10% FBS in PBS, incubated with anti-SOD3 antibodies (Affinity Biosciences, Cat# DF7753, RRID: AB2841219), incubated with a corresponding secondary antibody, and then stained with 3,3-diaminobenzidine.

Techniques: In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: SOD3 suppresses early cellular immune responses to parasite infection

doi: 10.1038/s41467-024-49348-0

Figure Lengend Snippet: a – d Statistical analysis of CD11b hi F4/80 + monocyte-derived macrophages ( a ), neutrophils ( b ), monocytes ( c ), and CD11b low F4/80 + macrophages ( d ) from WT mice at different time points after P. berghei ANKA infection. n = 7-8 (a-d) Multiple comparisons test corrected for Tukey was used to test for significant differences between uninfected and infected groups. e , f Transcription of the gene coding for SOD3 was determined by RT–qPCR in neutrophils ( e ) and macrophages ( f ) after parasite infection ( n = 3). An ~10-fold increase in gene transcription was observed in neutrophils but not in macrophages. Statistical significance tested by one-way ANOVA, and multiple comparisons to the uninfected control were corrected using Dunnet’s method. Three biological replicates were included in the experiment. g Representative high-magnification images of spleen sections of WT mice analyzed with double immunofluorescence for SOD3 (red) and myeloperoxidase (MPO, green). Significant augment of SOD3 expression in the splenic tissue of infected mice was observed. Arrow heads indicate representative cells with strong colocalization. Three biological replicates were included in the experiment. Scale bar =20 µm. h Comparable Western blotting analysis of SOD3 expression in neutrophils sorted from uninfected or infected mice. SOD3 expression in neutrophils of infected mice was significantly more than that from uninfected mice. Three biological replicates were included in the experiment. Each experiment was repeated three times independently with similar results. The neutrophils illustration was created with http://BioRender.com (publishing license: TB26UIITUU). i Schematic representation of the procedure for adoptive transfer. The illustration was created with http://BioRender.com (publishing license: NC26UIHFCR). j Adoptive transfer of WT neutrophils into SOD3 −/− mice resulted in early death of SOD3 −/− mice after P. berghei ANKA infection. Five biological replicates were included in the analysis. Kaplan‒Meier survival curves were calculated using the survival time for each mouse in all groups, and significance was determined by the log-rank test. Histograms present the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Brain sections were blocked with 10% FBS in PBS, incubated with anti-SOD3 antibodies (Affinity Biosciences, Cat# DF7753, RRID: AB2841219), incubated with a corresponding secondary antibody, and then stained with 3,3-diaminobenzidine.

Techniques: Derivative Assay, Infection, Quantitative RT-PCR, Control, Immunofluorescence, Expressing, Western Blot, Adoptive Transfer Assay

Journal: Scientific Reports

Article Title: A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge

doi: 10.1038/srep24082

Figure Lengend Snippet: Thirty larvae were injected each with 200 ng of PcSOD3.1 -, PcSOD3.2-, PcSOD1- dsRNA or gfp -dsRNA. Five days after this initial injection, larvae were challenged with microbes. The probability of survival is depicted as Kaplan-Meier curves by applying Gehan-Breslow statistic test. The weight of freshly emerged pupa was determined after RNAi silencing and infection of larvae. ( A ) Survival upon fungal challenge. After emptying of the defence reservoirs, the larvae of each group were submerged at the same time in the same solution of M. anisopliae 1*10 6 conidia/ml. Overall level of significance: p = 0.014 (Gehan-Breslow test); p = 0.04 (Log Rank (Mantel-Cox)). Pairwise multiple comparison procedures for gfp vs. Pc SOD3.1(−): p = 0.04 (Holm-Sidak method). Pupal weight, level of significance: p = 0.023* (two-tailed t-test). ( B ) Survival after injection of 1*10 6 E. coli -cells/200 nl. ( C ) Survival after injection of 1*10 6 M. luteus -cells/200 nl.

Article Snippet: Using the Calbiochem Autoinduction system 1 (Merck, Darmstadt, Germany), recombinant (r) Pc SOD1, r Pc SOD3.1, and r Pc SOD3.2 were produced in E. coli strain BL21(DE3)star (Invitrogen, Thermo scientific) and purified according to Frick et al. . A negative control has been included with a recycled pET100-TOPO vector with no insert.

Techniques: Injection, Infection, Two Tailed Test

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: SOD3 is released from a preformed pool. HMDMs were stimulated by the addition of LPS and the supernatant collected at the indicated time points. The supernatant and corresponding cell lysates were analyzed by (A) immunoprecipitation/Western blotting. As a control of immunoprecipitation, we performed parallel analysis in the absence (−) or presence (+) of purified recombinant SOD3. Additionally, the presence of SOD3 in the supernatant was evaluated by ELISA (B). (C) The transcriptional level of SOD3 upon LPS stimulation was evaluated by RT-qPCR and presented relatively to the level in the absence of LPS (t = 0 min). This analysis shows that the release of SOD3 from macrophages is relative fast and does not reflect a transcriptional upregulation. Error bars in panel B and C represents mean ± SD ( n = 3).

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Immunoprecipitation, Western Blot, Control, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: LPS-stimulation mobilize SOD3-containing intracellular vesicles. (A) HMDMs were pre-incubated with BAPTA-AM at the indicated concentrations and SOD3 release subsequently stimulated by the addition of LPS (100 ng/ml). Cell culture supernatants and lysates were analyzed for the presence of SOD3 by immunoprecipitation and SDS-PAGE/Western blotting. Control for immunoprecipitation is indicated on the left. (B) The level of SOD3 in the cell culture supernatant was likewise determined by using ELISA. (C) Immunofluorescence of unstimulated HDMDs shows an intracellular localization of SOD3 in small vesicles, that does not overlap with localization of lysosomal markers AP3δ, LAMP1 or DiI-conjugated Ac-LDL. Images are representative of three separate experiments. These data show that SOD3 is mobilized from stimulated macrophages by exocytosis of intracellular vesicles.

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Incubation, Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: Cell surface-associated SOD3 in increased upon stimulation. (A) The level of cell surface-associated SOD3 on resting and LPS-stimulated HMDMs was evaluated by using flow cytometry. Stimulated cells were fixed after the indicated time points and surface associated SOD3 detected by using an APC-conjugated mAb 5G8D4. Cells were gated to select live and monodisperse HMDMs . The analysis was repeated three times with cells obtained from separate donors producing similar results. (B) Cell surface-associated SOD3 was evaluated by confocal microscopy. Cell surface staining of SOD3 and cholera toxin B-subunit (lipid rafts) on live HDMDs treated or not with LPS shows a substantially increased localization of SOD3 to the surface of LPS-treated cells. These analyses show that the level of cell surface-associated SOD3 is increased by LPS stimulation.

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Flow Cytometry, Confocal Microscopy, Staining

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: SOD3 is internalized and stored intracellularly. (A) HMDMs were added medium containing ovalbumin or cMyc-tagged SOD3 and incubated for 90 min. The medium was subsequently removed, the cells washed in the presence of heparin (50 U/ml) and cells subsequently lysed at the indicated time points. The recovered lysates were analyzed for the presence of ovalbumin and SOD3-cMyc by SDS-PAGE/Western blotting. The analysis was performed three times. This analysis shows that internalized SOD3-cMyc is maintained within the cell whereas ovalbumin is degraded.

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Incubation, SDS Page, Western Blot

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: Internalized SOD3 is released from stimulated HMDMs. Cells were cultured in the absence or presence of SOD3-cMyc for 90 min to allow for internalization. Cells were washed and added buffer or stimulated by the addition of LPS for 1 h as indicated. Cell culture supernatants were collected and analyzed by immunoprecipitation and SDS-PAGE/Western blotting. Two separate membranes were developed using an anti-SOD3 antiserum and Mab 9E10 anti -cMyc. Purified SOD3 and SOD3-cMyc was included as controls on the left. This analysis shows that internalized SOD3 is released from HMDMs upon LPS stimulation.

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Purification

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: SOD3 is internalized by receptor-mediated endocytosis. (A) HMDMs were incubated with SOD3-cMyc (500 ng/ml) in the presence of increasing amounts of heparin as indicated for 90 min. Cells were subsequently washed, lysed and the recovered lysate analyzed by immunoprecipitation and SDS-PAGE/Western blotting. The uptake of SOD3 was evaluated by using the mAb 9E10 anti -cMyc. (B) The involvement of LRP1 in internalization was evaluated by incubating SOD3-cMyc in the presence of increasing molar ratios of RAP, an inhibitor of LRP1-ligand interactions. The cells were incubated in 90 min and the lysates analyzed as in (A). The experiments were repeated three times with similar results. This analysis shows that SOD3 is internalized into HMDMs via LRP1 interaction.

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Incubation, Immunoprecipitation, SDS Page, Western Blot

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: SOD3 modulates the pro-inflammatory cytokine profile of LPS-stimulated macrophages. Bone marrow-derived macrophages were established from wild-type and KO mice and stimulated by IFNγ and LPS for 16 h. Cell culture supernatants were collected and the pro-inflammatory cytokine profile was established by using the Mouse cytokine array panel A (R&D systems). Cell culture supernatants were pooled from wild-type mice ( n = 3) or SOD3 KO mice ( n = 3) and used to probe the membrane. The intensity of the developed spots was evaluated as described by the manufacturer by using ImageJ software and presented as corrected pixel intensity with error bars indicating mean ± SD. Data were analyzed by Student's t-test and p < 0.05 indicated (*).

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Derivative Assay, Cell Culture, Membrane, Software

Journal: Redox Biology

Article Title: The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

doi: 10.1016/j.redox.2019.101268

Figure Lengend Snippet: Schematic showing the cellular cycle of SOD3 in macrophages. The secreted protein binds to glycosaminoglycans on the cell surface and is internalized via the interaction with LRP1 (1). The internalized SOD3 is sorted and stored in intracellular vesicles of unknown identity (2). Upon stimulation, the macrophage has the capacity to mobilize SOD3 from these vesicles to allow for an acute increase at the cell surface as well as in the surrounding tissue (3). The concomitant expression of NOX2 activity supports the generation of superoxide (O 2 .- ) (4). The increased SOD3 activity in the extracellular space has the capacity to modulate the redox environment and affect endocrine and paracrine redox-dependent signaling by generating H 2 O 2 as a secondary messenger (5).

Article Snippet: The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose.

Techniques: Expressing, Activity Assay